Development of a Comprehensive Mutational Pannel as an Effective Tool for Personalized Diagnostic of Medullary Thyroid Carcinomas

 

Medullary Thyroid Carcinoma (MTC) originates from mutations in calcitonin-producing parafollicular C cells of the thyroid, is a rare malignancy, accounting for 3-4% of all thyroid carcinomas. It occurs in a hereditary form (HMTC, 25%) or in a sporadic form (SMTC, 75%). The prognosis for patients with MTC is poor, as the tumor metastasizes at early stages; and the only curative therapeutic option so far is radical surgery. Genetic analysis helps identify inherited cases at a stage where prophylactic surgery can be offered to carriers of such mutations to prevent the disease. This approach may also be used to determine better treatment options for patients who are already diagnosed with MTC.

The goal of this project was to develop a comprehensive mutational panel for the detection of clinically relevant mutations in MTC samples. A total of 143 mutations (nucleic acid variations) in 8 human genes were selected from numerous papers and public databases and included into the MTC mutational panel. The selection criteria were based on the coding mutations (mutations that occur in the coding area of the genes) that were reported to occur in MTC patients and considered functionally relevant. Some of the mutations included into the MTC mutational panel (such as BRAF gene V600E mutation) were also described in other types of thyroid cancer, but most of the mutations were unique to MTC (such as all RET gene mutations). The entire assay design was carried out using Sequenom's online design tools (ProxSNP and PreXTEND (https://www.mysequenom.com/Tools) and Assay Design software (v. 3.1)).

The final file comprised from 115 assays corresponding to all 143 mutations included into the MTC panel. This file will be further processed using the SEQUENOM® Mass-ARRAY iPLEX® platform for DNA genotyping of clinical samples by the cancer research scientists at the Abramson Cancer Center of the University of Pennsylvania.

 

References:

1. Millis, M. (2011, Summer). Medium-Throughput SNP Genotyping Using Mass Spectrometry: Multiplex SNP Genotyping Using the iPLEX® Gold Assay. Springer Protocols, 700. Retrieved August 20, 2012, from http://link.springer.com/protocol/10.1007%2F978-1-61737-954-3_5

This paper discusses and explains the basics of genotyping with the using MALDI-TOF Mass Spectrometry. This was the first paper that I have read right after being given the project. After fully interpreting this paper, I realized that I am going to be able to finish the project on my own. This paper served as a guide to me throughout the process of doing this project, as well as while writing the entire research report. In addition, this paper was used to make sure that I am not saying something that is factually incorrect.

 

2. Gabriel, S., Ziaugra, L., & Tabbaa, D. (2009, January 1). UNIT 2.12 SNP Genotyping Using the Sequenom MassARRAY iPLEX Platform. Current Protocols in Human Genetics.

This paper describes in details the SNP genotyping method based on the Sequenom MassARRAY platform. It includes two step protocol (initial locus-specific PCR reaction, followed by single base extension using mass-modified dideoxynucleotide terminators) an assay structure and how using MALDI-TOF mass spectrometry identify the SNP allele. The paper is mentioned in the corresponded section of the Introduction.

 

 

3.      Ricarte-Filho, J., Ryder, M., Ghossein, R., Fagin, J., Chitale, D., Rivera, M., et al. (2009, June 1). Mutational Profile of Advanced Primary and Metastatic Radioactive Iodine-Refractory Thyroid Cancers Reveals Distinct Pathogenetic Roles for BRAF, PIK3CA, and AKT1. CANCER RESEARCH. Retrieved July 1, 2012, from

http://cancerres.aacrjournals.org/content/69

The paper describes profiling of 111 mutations in RET, BRAF, NRAS, HRAS, KRAS, PIK3CA, AKT1 genes in clinical poorly differentiated, anaplastic and radioactive iodine-refractory differentiated thyroid cancers. The genotyping method is based on the Sequenom MassARRAY platform. It was shown that RAS mutations were prevalent in primary PDTC, whereas BRAF was more common in metastatic PDTC and ATC. PIK3CA or AKT1 mutations were rare. The paper is mentioned in the corresponded section of the Introduction.

 

4. MEN2 Database. (n.d.). AURP Scientific Resource for Research and Education.. Retrieved July 12, 2012, from http://arup.utah.edu/database/MEN2/MEN2_display.php

This database was only used for its mutations list during the collection phase of the project.

 

5. Human BLAT Search. (n.d.). UCSC Genome Browser. Retrieved August 17, 2012, from http://genome.ucsc.edu/cgi-bin/hgBlat

The Human BLAT database was used for alignment of nucleic sequences. The sequences retrieved from COSMIC were inputted in the BLAT Database to be aligned with the rest of the nucleic sequence, as only a small part of it could be gathered from COSMIC.

 

6. Catalogue of Somatic Mutations in Cancer - COSMIC. (n.d.). Wellcome Trust Sanger Institute. Retrieved July 10, 2012, from http://www.sanger.ac.uk/genetics/CGP/cosmic/

This database was used to retrieve the nucleic sequences that were used as the base to be inputted in the BLAT database. As it is impossible to use BLAT database with only knowing the position of the mutation, COSMIC was used to retrieve the minimal part of the sequence required to find the full nucleic sequence for any particular gene.

 

7. Genetics of Endocrine and Neuroendocrine Neoplasias (PDQ®). (n.d.). National Cancer Institute. Retrieved July 12, 2012, from http://www.cancer.gov/cancertopics/pdq/genetics/medullarythyroid/HealthProfessional/Table4

This database was only used for its mutations list during the collection phase of the project.

 

8. OMIM Entry - # 171400 - MULTIPLE ENDOCRINE NEOPLASIA, TYPE IIA; MEN2A . (n.d.). OMIM - Online Mendelian Inheritance in Man . Retrieved July 19, 2012, from http://omim.org/entry/171400

This entry was used as the basis of understanding of the MEN 2A syndrome. It talks about the relationship between MTC and diseases such as pheochromocytoma, and about the basics of what MEN 2A syndrome actually is. In addition, it talks about what mutations are associated with the syndrome. It was specifically used for comparison with the MEN 2B and FMTC.

 

9. Jimenez, C., Hu, M. I., & Gagel, R. (2008, Spring). Management of Medullary Thyroid Carcinoma. Elsevier Saunders, ?, 15.

This MTC review was the first of many that I have read in the duration of this project. This review provided me with the basic information about MTC without which any attempt at actually finishing this project would have been obsolete. Many parts of the introduction are referred to this paper, as it was very influential. Similarly to many other sources, mutations were taken from this publication during the first phase of the project.

 

10. MacConaill, L Profiling Critical Cancer Gene Mutations in Clinical Tumor Samples. PLoS ONE (2009). http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007887

This publication was one of many that were used only for its mutations. No part, except for the abstract, which contributed to the overall idea of the research report, has been read.

 

11. Ehsan Alvandi, Seyed Mohammad Akrami, Mohsen Chiani, Mehdi Hedayati, Babak Noori Nayer, Mohammad Reza Mohajeri Tehrani,et al. (2011, April 5). Molecular Analysis of the RET Proto-Oncogene Key Exons in Patients with Medullary Thyroid Carcinoma: A Comprehensive Study of the Iranian Population. Thyroid, 1. Retrieved September 1, 2012, from http://online.liebertpub.com/doi/abs/10.10

This publication was one of many that were used only for its mutations, and or one small piece of information. No part, except for the abstract, which contributed to the overall idea of the research report, has been read.

 

12. Moura, M., Cavaco, B., Pinto, A., & Leite, V. (2011, February 16). High Prevalence of RAS Mutations in RET-Negative Sporadic Medullary Thyroid Carcinomas. JCEM ONLINE, 95, 6.

This paper has shown a study where 64% of the patients that had Sporadic MTC were found to have a BRAF mutation in position 600. This is very unusual as this mutation is considered to be PTC specific. In addition, this publication has given additional context to this research report. Similarly to many other sources, mutations were taken from this publication during the first phase of the project.

 

13. OMIM Entry - # 162300 - MULTIPLE ENDOCRINE NEOPLASIA, TYPE IIB; MEN2B . (n.d.). OMIM - Online Mendelian Inheritance in Man . Retrieved June 19, 2012, from http://omim.org/entry/162300 

This entry was used as the basis of understanding of the MEN 2B syndrome. It talks about the relationship between MTC and diseases such as pheochromocytoma, and about the basics of what MEN 2B syndrome actually is. In addition, this entry talks about what mutations are associated with the syndrome. It was specifically used for comparison with the MEN 2A and FMTC.

 

14. hybridization, f. i., & (1989), I. e. (n.d.). OMIM Entry - + 164761 - REARRANGED DURING TRANSFECTION PROTOONCOGENE; RET . OMIM - Online Mendelian Inheritance in Man . Retrieved July 19, 2012, from http://omim.org/entry/164761

This entry was used as the basis of understanding MTC, and why the mutations RET gene are found in so many cases of MTC. Unfortunately, as with many other publications, no definitive answer was given. This entry was also used as a guide, to make sure that what I say about mutations in RET gene is factually correct.

 

15. RT-PCR., & (2003), M. e. (n.d.). OMIM Entry - # 155240 - THYROID CARCINOMA, FAMILIAL MEDULLARY; MTC . OMIM - Online Mendelian Inheritance in Man. Retrieved July 19, 2012, from http://omim.org/entry/155240

This entry was used as the basis of understanding of the FMTC syndrome. It talks about the relationship between MTC and diseases such as pheochromocytoma, and about the basics of what FMTC syndrome actually is. In addition, this entry talks about what mutations are associated with the syndrome. It was specifically used for comparison with the MEN 2B and MEN 2A.

 

16. Hazard, J., Hawk, W., & Crile, G. (1959, January 1). MEDULLARY (SOLID) CARCINOMA OF THE THYROID—A CLINICOPATHOLOGIC ENTITY. JCEM, 19. Retrieved June 26, 2012, from http://jcem.endojournals.org/content/19/1/152

This publication was the first time MTC was classified. The paper itself was not read by this applicant, however, due to its historic relevance, it was referred to in the first paragraph of the introduction.

 

 

17. Cakir, M., & Grossman, A. (2009, May 25). Medullary Thyroid Cancer: Molecular Biology and Novel Molecular Therapies. Neuro Endocrinology, 25.

This publication, alongside many MTC reviews, was used as the guideline for this research report. Many facts, such as information about Sporadic and Hereditary MTC were confirmed by this publication. Similarly to many other sources, mutations were taken from this publication during the first phase of the project.

Process Paper:

For this project I wanted to test my limits as a director, actor, writer and camera worker. I wanted to emphasize my leadership skills in a way that would not only help myself but help and benefit others and those around me. When this project started I had many visions that would and would not come to pass, but I never let that stop me. During the capstone process I created and designed my own production company with my own notorious crew of misfits making films and learning how to better our skills. We started out sophomore year as some friends with basic knowledge of film and the heart to keep learning. By the time I got to senior year I was looked up to in film by a lot of the under class intro students, so i took it upon myself as part of representing Bad Habbit' to SAT in my spare time for Herman's digital video intro class. I try to teach even the kids that aren't in my ILP that I instruct. With that being addressed I have strained to teach anyone who needs film help despite not having the time or energy to do so. I have been working hard to make films by writing, directing, filming and acting with Jesús, Joseph, Steven and Jenny. Aside from teaching, learning and making films we also wanted to try being more professional and scored ourselves our first film job where we filmed a party event for a company that holds parties and vacations for the mentally disabled. We filmed the whole event and edited a video for them to use and hand out to their customers and future customers. This gave me a lot of confidence in film and was something that made me strive even more for my future film career. 

    In the end I chose this as my capstone because it was a huge part of my life and my life as a student as SLA, without which I would never have started on the path of film. The project just sort of spoke to me, I wanted to teach and learn and make films that I could show people and be proud of. When i set out to do this project all I wanted was recognition for not just myself, but also for my crew. I wanted us to be known as the kids who were good with film and could help teach and train others to do the same. I chose this because it worked and was something I had been working on extremely hard and making it my capstone gave me more room to work on it and make it an even bigger thing that what it was when we started. The goal of this for me was to simply make something out of nothing and create something I was proud of. This project was not easy we ran into fights and other issues along the way, but we all came out of it having worked hard and created something great that we plan to continue to work on both in and out of SLA with and without rough cut through out our time together over the next few years. 

When it comes to the core values of SLA I think my capstone did a good job of covering all of them at once. Inquiry in our ability to strive to continue to learn film throughout the whole process, Research in the loads of reading, watching videos and learning hands on about film and the things i searched for for inspiration, Collaboration is evident from the shared capstone I had with Jesús as well as the students we worked with and taught, Presentation in the fact that we created videos that were presented to the public and shared all over social media and reflection in the fact that after we finished each film we talked about what we could improve or innovate so when we went to film our next work it would be better and would show that we are also getting better. This capstone was for us and also the students of SLA. 

When we set about creating this capstone I had already been working with the ILP and making films for two years so I had a very good idea of what I wanted to do. First was our company workers. I had to recruit a crew and that was going to be simple. I had Joe Tartaglia as my co-director, Jesús as my partner/ co-founder as well as chief editor, Jenny Cruz as my make-up and costume design and Steven Webb as one of my star actors as well as various extras. My next step was research for inspiration and skills. When inspiration struck Jesús and I would sit down and design a story and script after we finished this process we brought it to the crew and got ready to create our film we then went through pre-production and production phases. During post production jesús and I would sit down to edit our footage. Once we had a final product I would upload it to youtube.com Facebook as well as various other social media. This was our process through the year and on the side i would teach small lessons for choreography and film work to the crew so they knew what they were doing as well. This was a process throughout the year.

Throughout this process we encountered a lot of obstacles the ones that really hindered us were the amount of school work we had making a imbalance in time to work on the project. We also had an issue with teenage drama that did make for some very difficult working conditions. We also had a problem with technical issues that put us behind for time. We needed to fix these issue unfortunately we couldn't stop the course load of work so we decided to incorporate our digital video assignments with our company so we could have access to more people and better ideas. When it came down to drama we had to replace certain people or let people leave temporarily until things were better and finally for the technical issue we had to back up our files better and put a better lock down on our software and footage to keep it safe. 

When i was trying to complete my capstone I had to work hard and find lots of different things to help me as I went along. This was hard because I hate working really hard and its not always easy to find the right resources. I had to find something to help me with ideas and motivate me to keep doing what I was doing and although I found a bunch of small stuff along the way it seemed nearly impossible to find it. That s when it finally hit me and I had realized my best resource was right under my nose the whole time. I loved to watch Rooster Teeth Productions' videos and CG work as well as their podcasts and buy their merchandise. They were the perfect example of what I wanted our company to be like. They started as a bunch of video game playing friends that got together and made films using their video games and from there they went on to become famous and sell tons of their stuff as well as please a giant audience of viewers. I researched everything that they did over time so I could stay inspired and continue to aspire. 

The things I am most proud of about this capstone are my fellow students that I watched work super hard and get better and better as the year went by. This made me feel good about myself and the project because the thing I really cared about in this project was to make film and to help those around me do so as well. I learned so much from this process about myself and film and art as well as that with work, friendship and the mindset to do something anything is possible.  If I could do this project again I would have strived to not let drama get into the way of our work and I would have tried harder to please those around me so they had a easier fun time with the film we produced. SLA may not exactly be a better place because of me but I feel as if i brought at least some type of memorable flare to my work and to my friends around me and that is really all I care about. I hope that in doing this project I would have made more students comfortable making films and maybe even inspired some people to make films as much as I do. 

  

 Abstract:

  For this capstone I had a vision and I knew exactly what I wanted to do. Although a lot of things changed during this process the hopes and dreams of my peers and I never faded. We set out sophomore year as two art students with a bunch of ridiculous ideas in our heads, Jesús and I built the show Dylan's dumpster and not long after with it a free form digital video ILP. We created a show and built up a cast. Once I realized that I was capable of making something like that I set out on a career path that no once has been able to stop me in. I work hard to press my limits in film and created my best work possible. I decided to team up with Jesús for a new project and once we built Members Only productions, he left and I had to build a new cast. Joseph Tartaglia helped me achieve this goal and became my new partner. Together we created and whole new line of short films and trailers for our audience amusement. That was the entirety of my sophomore and junior year. Senior year I still had the same dream and Joe had become just as motivated as me. I wanted to make a capstone that would be inclusive and would produce great work. Together with Jesús and Joe I set out to make Bad Habbit' productions our new company. We started making some new films right away that grew from digital video and our ILP sessions with other students. We filmed our trailer for a zombie film called complication Z to start up our new year and that became a great success to us. We filmed all over the city in random woods and abandon train track to in houses and school. I pushed my limits with my ability to work the camera to almost a perfection. I even make sure as part of representing my company that I take away my x2 lunch on occasion to help SAT Mr. Herman's digital video intro class. I like to teach as well as work in every other field of the job so I took this year to take up everything at least once. I worked with make up, directing, acting, filming, editing, stunts, prop making, choreography and script writing. This was a challenge but I loved every minute of it and teaching it to those around me made me feel even better. That was what I set out to do for this capstone I wanted to teach film while at the same time prove that with help I can produce a whole lot of funny and credible films. I do have visions for the future of coming back to further help teach when I have free time and helping joe run Bad Habbit' from the outside while he keeps the ILP running for the underclassmen including Marshal woodruff who will possibly be our future chief editor to take over for jesús if he accepts our offer. We wanted him to feel welcome in a community of film students that want to make films with him as well as Joe who will be taking over for me and some other future juniors as well as hopefully some sophomores that may be interested. This as well as our outside business working professional jobs is off to a great start, but it is still only a start we have worked hard this year and I have strived to make this as perfect as I could and I am very proud of the work my team has made and of my capstone's message and affect in digital video classes. 

Capstone Products:

- ​Cordivari , Dylan, dir. Cray-4: Regaining the fame. Bad Habbit' Productions , 2012. Film.  May 2013. <https://www.youtube.com/watch?v=L9EqyXperkE>.

- Cordivari , Dylan, dir. Complication Z. Bad Habbit, 2012. Film.  May 2013. <https://www.youtube.com/watch?v=cu_bV25u2TY>.

- Cordivari , Dylan, dir. Grande getaways: Christmas Party. Bad Habbit, 2013. Film.  May 2013. <https://www.youtube.com/watch?v=LcskgtSF4Es>.

- Cordivari , Dylan, dir. Joe's Fantastic day: Part 2. Bad Habbit, 2013. Film.  ay 2013. <https://www.youtube.com/watch?v=_q7KhyJGZO4>.\

- Cordivari , Dylan, dir. A love found is a love lost. Bad Habbit, 2013. Film.  May 2013. <https://www.youtube.com/watch?v=jGhxXfu62VM>. 

- Cordivari , Dylan . "Bad Habbit's Productions channel."Itschannel69. Bad Habbit's Productions , n. d. Web. May. 2013. <https://www.youtube.com/user/ItsChannel69>.


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